Aim of this study was to evaluate the influence of normobaric hypoxia on myocardial function in healthy humans. Fourteen subjects underwent two-dimensional speckle tracking echocardiography (2D-STE) examination during normoxia and in a normobaric hypoxia chamber. Examinations were performed at rest and during bicycle exercise test. The following parameters were quantified in both atria and ventricles by 2D-STE: Global Strain (S), systolic strain rate (SR(S)), early (SR(E)) and late (SR(A)) diastolic strain rate. During hypoxia SR(S) and SR(E) increased significantly in both ventricles compared to baseline. The increase of LV SR(S) and SR(E) during normoxic exercise was significantly higher when compared with exercise under hypoxia (for SR(S) -0.55 ± 0.22 vs. -0.34 ± 0.24 1/s, p = 0.024; for SR(E) 0.56 ± 0.29 vs. 0.23 ± 0.29 1/s, p = 0.005). For the right ventricle (RV) no significant difference of exercise induced increase of systolic contractility was found (SR(S) -1.07 ± 0.53 under normoxia vs. -1.28 ± 0.24 1/s under hypoxic conditions, p = 0.47). A shift from passive conduit (SR(E)) to active contraction (SR(A)) phase during hypoxia was noted for the right atrium (RA) (SR(E)/SR(A) 0.72 ± 0.13 under hypoxia vs. 1.17 ± 0.17 under normoxia). The ratio SR(E)/SR(A) of RA was closely related to pulmonary systolic pressure (r = -0.78, p < 0.001). Exposure to normobaric hypoxia leads to an increase of regional myocardial deformation in both ventricles. The contractile reserve during hypoxic exercise is reduced in LV, whereas RV systolic deformation rate is maintained. In addition, hypoxia had an impact on the ratio of passive conduit to active contraction phase in right atrium.